EP3793720A1

EP3793720A1 - Microarray transformer

Info

Publication number: EP3793720A1
Application number: EP19724814.9A
Authority: EP
Inventors: Günter Roth; Stefan Daniel KRÄMER; Johannes Wöhrle
Original assignee: Biocopy GmbH
Current assignee: Biocopy GmbH
Priority date: 2018-05-15
Filing date: 2019-05-15
Publication date: 2021-03-24
Also published as: KR20210006976A; WO2019219757A1; CN112218708B; CN112218708A; US20210213414A1

Abstract

The invention relates to a method for microarray transformation, in which an original array can be copied onto a planar carrier by using a cavity chip having a transformation matrix, and the information or spatial arrangement is changed in such a way that a transformed second array is produced. The invention further relates to a device for carrying out such a method.

Description

Microarray transformer

Summary

The invention relates to a method for microarray transformation in which, by using a transformation matrix cavity chip, a template array can be copied onto a planar support and the information or spatial arrangement is thereby changed so that a transformed second array results. The invention also relates to a device for carrying out such a method.

State of the art

DNA microarrays are an accumulation of many different, small dots (spots) with DNA on a solid substrate. In the production of DNA microarrays basically a distinction is made between three different types of production:

1. Spotted DNA Microarrays a. Microarray spotter [8]

2. In situ synthesized DNA microarrays a. Spot synthesis; Inkjet printing [2], [3] b. Photolithography using photomasks [10] c. Photolithography by means of micromirrors [9]

3. Synthesis by DNA polymerase

A relatively novel method for the production of DNA microarrays is to synthesize the DNA on the surface by means of a polymerase using a DNA template (W02009034181A2, W02010100265A1). This is a fixed

Surface provided with so-called primers (synthesis starting points for the DNA polymerase). Subsequently, a mix consisting of the individual synthesis building blocks, the DNA polymerase and the template is added to this very surface. The synthesis is highly parallel up to several thousand points. The reaction space of each of these points was physically separated to ensure an independent synthesis reaction. This can represent a spatial separation over microcavities to the limitation of the diffusion.

The fundamental difference in the methods of preparation is that the DNA molecules are prepared for (1) in advance and for (2 and 3) during the creation of the DNA microarray. In all the above-mentioned production techniques for DNA microarrays, the individual arrays are each newly manufactured, ie completely from the front. For this reason, there are several approaches that aim to replicate existing DNA microarrays:

The duplication of DNA microarrays by hybridization:

Here, complementary DNA strands from DNA pools are hybridized to an existing DNA microarray. The complementary DNA molecules each have a reactive group with which it is possible to attach them to a second substrate in which both are brought into contact. This substrate now contains the complementary DNA molecules of the original DNA microarray and has the same local resolution. A copy produced in this way is comparable to the negative of an analogous photograph ([4], [5], [6], [1 1], [12], US20060141245A1),

W020061 12815A2).

Reproduction of DNA microarrays by hybridization and extension by DNA polymerase:

This method is very similar to the previously described method. However, the hybridized DNA molecules are significantly shorter than the DNA molecules of the DNA microarray to be amplified. The hybridized DNA molecules in this case serve as primers for a DNA extension reaction using DNA polymerase. The polymerase is an enzyme that can extend a DNA strand based on a template accordingly. By means of this method it is in principle possible to duplicate DNA microarrays with long DNA molecules. A copy produced in this way is comparable to the negative of an analogous photograph ([6]),

US20100256017A1, W02008022332A2).

Duplication by means of a master cavity chip and a subsequent PCR

A master cavity chip has DNA primers on its surface that are complementary to the DNA of an existing DNA microarray. This chip is filled with PCR mix and placed on the DNA microarray to be replicated. By means of a PCR, the information of the DNA of the DNA microarray is transferred into the master cavity chip and stored. In a second step, the cavity chip is filled again with PCR mix and closed with an empty microarray. Subsequently, another PCR is performed to transfer the DNA molecules from the master cavity chip to the new surface. This thus represents an exact replica of the original DNA microarray. (W02010100265A1)

There is no method in the art for replicating and transforming existing DNA microarrays as desired.

The different methods of the prior art have various disadvantages:

Classic production methods (Array production): No replication of DNA microarrays is possible. Created, generated microarrays can no longer be modified / transformed using these methods. They would have to be generated from scratch. Subsequent transformation is therefore not possible. Spot size and shape is determined by the respective production and can not be chosen arbitrarily. The arrangement of the spots is also limited by the production method. No transformation of a DNA microarray is possible with any of the aforementioned methods.

Existing methods for the replication of existing DNA microarrays:

It is not possible to change the spot sizes or their specific locations (none

Space transformations). Likewise, no shape transformations are possible because the

Output array always determines the shape of the spots and determined. Besides, they are

Sequence transformations only possible in the form of extension or shortening of the existing DNA. Further transformations were not described.

Sequence transformations in the form of merging or combinatorics are not possible with existing methods.

In the prior art, an array is basically printed by a printer during a copying process. For each different DNA spot, a single DNA sample must be used, which requires a lengthy preparation. This makes the process time consuming and costly. In addition, lengthy washing between printing operations is necessary to prevent and reduce contamination between spots. Furthermore, it is necessary that the spacing of the spots is chosen to be sufficiently large to prevent running into one another and contamination.

Another disadvantage of the prior art methods is that the shape of the spots is very limited and only near-round spots are possible.

The drying of the liquid spots has an effect on the homogeneity of the spots, so that there is a difference in the result between the edge area and the center of the spot.

light synthesis

An array is made by photolithography. This results in the following problems.

Each step of the synthesis requires a single mask and extends the DNA strand by only one base. There is a certain percentage of errors per step. As a result, the length of the DNA strand affects the quality of the result. After each step of the synthesis, a washing step must be performed and the mask replaced, making the method time-consuming and costly. The design of an array can only be costly and costly

Time is changed. Maskless lithography with micromirrors, as with a projector / projector, makes the use of different masks obsolete, but this limits the shape of the spots to rectangular or square shapes

Solid Phase PCR

DNA is distributed digitally and randomized in a glass cavity chip and amplified by PCR. The production of the glass cavity chips is very complicated, and the patterns and spot shapes are limited to round and hexagonal arrangements. Furthermore, several different cavity shapes on an array are not possible. Operations such as zooming in and out are not possible with this method. It is also not possible to move spots or spot merging. A transformation of arrays is therefore excluded.

So far, no method is known or described in the prior art, with the aid of which DNA microarrays can be transformed. Only types of replication have been described. However, the transformation of microarrays opens up unanticipated and unexpected possibilities, e.g. the production of completely new DNA arrays.

The challenge or the task here is to get the information of the original one

To store and modify microarrays. While storing mostly good too

accomplish, the accompanying transformation is a big task.

Microarrays of the prior art can not be changed after their production, except possibly extend or shorten. However, this is often only possible to a limited extent due to the poor quality of the primary DNA. Furthermore, existing formats of microarrays can not be "reformatted" purely physical, i. you always have to adapt the experiments and devices to the existing microarrays or do without experiments if suitable microarrays are not available. In addition, two microarrays can not be "put together" into one after their production.

Description of the invention

The problem is solved by the independent claims. Advantageous embodiments emerge from the subclaims.

In a first preferred embodiment, the invention relates to a method of microarray transformation comprising the following steps a) providing a template array, wherein the template array comprises a plurality of spots with template molecules and wherein the template molecules are preferably oligonucleotides, b) providing a cavity chip comprising a transfer matrix, c) providing a reaction mixture in the cavity chip, d) placing the precursor array on the cavity chip, e) copying process, wherein the oligonucleotides of the spots of the precursor array are copied onto the cavity chip f) providing an array surface. g) providing a reaction mix in the cavity chip, h) placing the array surface on the cavity chip, i) copying process, whereby the oligonucleotides of the spots of the cavity chip are copied onto the array surface as DNA, RNA or protein and wherein this newly formed further array is in terms of spot shape, Spot size, spot location and / or in terms of containing information differs from the template array.

The invention thus provides a microarray which, in comparison to a

Output array is transformed.

Transfer matrix is the arrangement of the cavities and their design as well as their properties.

It may be preferred that the reaction mixture and / or other reaction components are precoated in the cavity chip.

In order to transform DNA microarrays, a special method was developed in which the replication and transformation can take place in one or more steps.

Special cavity chips can be generated with any shape. These include small cavities with a specific shape in which the template molecules of the original microarray can be stored.

"Stored" in this sense means that the information of the template molecules has been permanently transferred. This can be done in the form of 1: 1 copies of the template molecules. But also modifications are possible, for example RNA to cDNA, wherein the sequence information is retained. It is also possible that the original template molecules or a part thereof are transferred to the cavity chip.

It is preferred that an original oligonucleotide microarray (particularly preferably a DNA microarray) having a geometry 1, is transferred into a cavity chip with a geometry 2. The transmission can preferably take place by means of a PCR. But here are others too Methods with which biomolecules, such as DNA can be amplified, for example, isothermal DNA amplification, HDA, RPA or LAMP possible. The choice of primers during this transfer reaction, which is also referred to as copying process in the context of the invention, also determines which spots of the original arrays are transmitted. Thereafter, the geometry 2 of the cavity chip can be transferred to a planar support, also referred to as an array surface, whereby a second-order microarray is formed. Now another transformation step can be carried out, into a cavity chip of geometry 3, whereby a 3rd order array is created. In principle, it is possible to reorder the original microarray in any order step by step.

For the purposes of the invention, "copying" means both the 1: 1 copy (that is, for example DNA to DNA) and also other methods in which the information is copied, but the product differs from the starting molecule, e.g. DNA to protein, RNA to cDNA, RNA to protein or DNA to RNA. A product molecule different from the parent molecule is also called a derivative. All derivatives and derivatives of the starting molecules are possible and the person skilled in the art knows how to prepare the respective product molecules.

The copying steps are preferably carried out by known means known in the art, but may also be replaced by alternative methods which produce the same or a similar biochemical reaction or amplification. It is preferred that an RNA polymerase is used for the conversion of DNA to RNA. A reverse transcriptase is preferably used for the conversion of RNA into cDNA. For the conversion of DNA and / or RNA into proteins are preferably cell-free in vitro

Transcriptional and / or translational mixes used. If DNA is to be copied to DNA, polymerases, RPA (Replication Protein A) or a system for helicase-dependent amplification (HDA) can be used.

A typical transformation process is preferably as follows:

1. Fill the cavity chip with a reaction mix, e.g. PCR mix or cell-free expression system

2. Placement of the original array on the cavity chip

3. Performing a copying reaction, e.g. a PCR

4. Wash and block

5. Optional: Modification of the template molecules in the cavities

6. Optional: Repeat steps 1-4 with the same cavity chip but with another original microarray 7. Fill the cavity chip with reaction mix and seal it with an empty array surface

8. Performing a copying reaction, e.g. a PCR

9. Wash and block the newly formed array and cavity chip

In particular, the optional steps can be combined as desired. The resulting shape of the resulting transformed spot is given here by the shape of the cavity of the cavity chip. In particular, the above-described process can also be used repeatedly and also again on already transformed arrays.

The spatial arrangement, e.g. the resulting pattern is preferably defined by the first copying step in the cavity chip. However, a modification of the molecules can also take place after that, so that a transformation of the information can take place at several points in time.

Also preferred is the method, wherein a modification, extension, shortening, derivatization and / or inversion of the molecules takes place in the cavities of the cavity chip and / or during the copying processes. Under derivatization are modifications of the

To understand starting molecules, the information e.g. a sequence information is retained. For the purposes of the invention, this also includes the translation of DNA or RNA into peptide sequences.

It is particularly preferred that steps a) to e) be repeated at least once, wherein the same cavity chip but another template array or the same template array is used in another or the same orientation.

If the same template array is copied several times into the same cavity chip, it is particularly advantageous for the template array and cavity chip to be in a different orientation with respect to the repetitions. This can e.g. done by rotation of the cavity chip. The orientation may also be consistent, e.g. to get synergistic effects.

It is particularly preferred that the copying step comprises an amplification step.

The amplification can be a PCR, an isothermal amplification or an RT-PCT. The amplification step may also describe a transcription or protein expression.

It is preferred that the reaction mix is a PCR mix, an isothermal amplification mix, a reverse transcription mix, a transcription mix, or a cell-free expression mix. It is also preferred that a protein synthesis takes place so that, for example, in the reaction mixture, enzymes are contained which transcribe DNA into RNA and translate RNA into protein.

It is preferred that a possible protein synthesis only takes place in a copying step from the cavity chip to an empty array surface.

Other suitable mixes may be used, the skilled person being able to select them without being inventive himself.

Particularly preferably, the template molecules are oligonucleotides, preferably DNA molecules or RNA molecules.

In addition, it is preferred that a plurality of spots of the template array are combined in the cavity chip and / or further array, so as to produce a mixture of template molecules or the molecules derived therefrom and / or to generate a DNA or RNA which contains a plurality of partial sequences or Derivatives of these partial sequences of the template molecules from the respective spots comprises.

It is further preferred that spots from at least two template arrays in the further array are merged so as to create a mixture of copies and / or to produce a copy comprising a plurality of subsequences of these partial sequences

Template molecules from the respective spots comprises.

In a further preferred embodiment, the invention relates to the method, wherein a spot of the template array in the further array is subdivided into a plurality of spots.

It is also preferred that additional DNA molecules be added in solution such that the molecules (template molecules or product molecules or an intermediate) are extended around this DNA sequence.

It is particularly preferred that the template molecules are DNA molecules that have all or part of short, identical DNA sequences of preferably 10-30 base pairs.

Method according to at least one of the preceding claims, wherein the cavities of the cavity chip are coated with primers.

The primers preferably carry an additional DNA sequence at the 3 'end or at the 5' end.

Preferably, the method, wherein the additional sequences as a barcode for a

Location information can be used and / or the primer can contain a sequence for T ranskription and / or cell-free synthesis It is preferred that the transformation is spatially and / or temporally limited. The spatial boundary is preferably given by the edge of the cavities. The time limit can be realized by different factors. Time-limited reaction conditions can be, for example, a temperature which is exceeded or fallen below, a pH which changes over time, the irradiation of light or electric fields.

The invention makes it possible to change existing microarrays in terms of their spatial structure. In particular with regard to the spot shape, spot size and spot location. It is possible to change the shape of the spots as you like, but also their position (up to the deletion of spots). This can find different applications depending on the geometry: a) In a transformation without relevant size changes, a spot is converted into a spot with approximately the same size, but the shape of the spot can be changed arbitrarily. Due to the transformation process extremely sharp edges are created and spots with previously impossible geometry can be created. b) When transforming into smaller sizes, a spot can turn into several small spots

Be "divided" whose shape can be arbitrary. c) When transforming into larger sizes, two or more spots or their DNA can be fused. Depending on the process selection and primer sequence, the DNA can be imaged in parallel or sequentially on the target spot. That the DNA may then end up being a mixture at the surface (several sequences side by side on the surface), or else a combination construct with one sequence attached to the other, thus also producing complete gene products.

By performing multiple transformations, a microarray can theoretically be reformatted, spots removed or added from other arrays, or DNA or other biomolecules shortened or lengthened. An advantage of the invention is that it is thereby possible to construct or reformulate gene constructs or gene libraries combinatorially.

It was completely surprising that it was recognized by the invention that the static form of the original microarray can be changed and reformatted by means of transformation via a cavity transfer. Analogous to a Fourier transformation, basically any geometry can be generated by the invention. Affine or partially affine mathematical mappings can now be applied to the geometry of an array and its biomolecules. Likewise, additions by DNA or other biomolecules may take place (eg, by ligation or prolonging amplification) as well as subtractions (eg, by PCR with a primer producing a truncated DNA product, or by a restriction enzyme which directs the DNA to a particular one Sequence cuts off). Thus, will The abstract and theoretical transformation of mathematics is now also possible for physical microarrays based on DNA or RNA.

The advantages of a first-order transformation (carrying out a first transformation) are versatile. Thus, e.g. a microarray in the desired spot shape be multiplied. Previous arrays are production-related "round". By the invention, any structures are possible. For example, an array of hexagons could be created in the first order (see also Fig. 1) and then converted back into circles in successive transformations. The same could also be shown the other way around. Furthermore, classically produced microarrays are always inhomogeneous within the single spot due to the manufacturing process. The transformation process of the invention, however, is homogeneous over the entire surface, resulting in very homogeneous spots with very precise and almost arbitrary geometry. An application is therefore in the quality improvement of arrays. This allows microarray manufacturers to produce a master array and then use the transformation to create high-quality and homogeneous arrays 1. To create order. The geometric quality of the master array is low, only the DNA quality high, resulting in arrays 1. Order of high quality and geometry or structure leads.

Another advantage is that multiple spots can be fused together to produce either a mixture of biomolecules such as DNA (2 or more species per spot) or a construct (e.g., fusion DNA) on the spot. A corresponding representation is shown in FIG.

Another advantage is that a spot can be split into several small spots. This allows more accurate measurements, higher homogeneity, better binding kinetics (according to Ekins, Ambient Analyte Theory) and thus a higher signal yield.

The transformation process requires only one cavity chip in addition to the output array, some reaction mix, e.g. PCR mix (usually between 5 and 10 pl) and a copy surface on which the 1st order array is applied. This makes the production more unique

Geometries (not yet technically feasible) possible. In addition, geometries that were hitherto limited in terms of technology can now be produced much more cost-effectively. Also, the production of a variety of replications is now easily possible. The reformatting of arbitrary geometries, which was not technically possible up to now, by means of geometric transformation corresponding to affine mapping in mathematics

(Shearing, stretching, rotation, shear stretching), or even just applying these operations to subregions or individual points, is another advantage of the invention, which allows a variety of possible applications.

It may also be preferred that the device comprises a set of transformation matrices in the form of cavity chips, which depending on the order of their application, an affine

Create an image of the array. This means, for example, that the device is diverse Single matrices (on corresponding cavity chips) may have, with one moves, another enlarged, the next reduced. It is also possible, for example, always everything to the same size enlarged / reduced, then moved and then everything is brought back to the old size. This set of transformation matrices is then a set of individual operations that can replace each other. As with mathematical affine mapping, that is, a point mirroring can also be done as a reflection followed by a 180 ° rotation.

It is also preferred that the transformation method be used to produce DNA, RNA or proteins. It is preferably possible to generate new DNA combinations.

In a further preferred embodiment, the invention relates to a device for carrying out a transformation according to the invention, comprising

A cavity chip comprising a transfer matrix,

• at least one template array and

• at least one array surface.

It is preferred that the transfer matrix spatially limits the reaction. This is done in particular by the cavities and their borders.

In a particularly preferred embodiment, the invention relates to a cavity chip for carrying out a transformation process of the invention.

The following specifications are particularly suitable for the cavity chips. These embodiments are applicable to both the claimed method and the claimed device.

It is particularly preferred that the cavities of the cavity chip comprise a volume of less than 100 nl. Particularly preferred are volumes of less than 50 nl, most preferably less than 20 nl. It was surprising that particularly good results could also be achieved with volumes in the picoliter range.

It will be understood by those skilled in the art that for certain applications, however, even larger cavities may be beneficial, e.g. Cells or tissues are used as starting material.

The shape of the cavities can be chosen arbitrarily. Round, hexagonal or triangular shapes are possible. In principle, however, any desired shape can be used.

The following specifications have achieved particularly good results in tests:

4000

Examples and figures

The invention will be explained below with reference to figures and examples without being restricted to them.

Example 1: Sequence, shape and / or space transformation a) ST Addition of different information per spot

Step 1 :

Two primary arrays form the initial position. Both arrays may have the same or different spot sizes, symmetries and spacings. The orientation of the DNA Molecules on the surface as well as the length of the DNA does not matter. The only common feature of both arrays is that all DNA molecules must have a similar, short homologous DNA sequence of about 10-30 base pairs, a so-called.

The overlapping area.

Step 2:

First, a scan of one of the primary arrays is made by PCR using a transmitter chip and a conventional PCR mix. After completion of the PCR, the same DNA species are present in the transmitter chip, which are also present on the starting DNA microarray. Their spatial arrangement has also been preserved. As a result, both the spatial, as well as the content information of the primary array was thus transferred to the Überträgerchip.

Step 3:

This transmitter chip is then placed on the second primary array. Again, a scan is performed using a standard PCR mix. Due to the homologous sequences of both original DNA microarrays, two scenarios are conceivable. If a DNA spot of the second microarray encounters an empty cavity, the DNA molecules are readily transferred into this cavity as in the first scan. However, if the DNA spot hits a cavity in which the DNA of the first starting array already exists, then both DNA strands are fused together on the cavity surface. New DNA molecules are created that carry both the information from the first and the second original DNA microarray.

Step 4:

The stored information in the form of fused DNA molecules or original DNA molecules of the transmitter chip can be copied by PCR and a standard PCR mix to a new, empty, specially functionalized surface. b) ST Addition of the same information per spot

With this step, identical DNA sections can be added to the surface or, in the case of a multiplicity of spots, or, if necessary, removed. The planar introduction can be done by an array which has significantly larger structures than the transfer matrix, or over the entire transfer matrix, when the DNA is coated around the e.g. is to be extended, brings directly into solution.

Step 1 :

A primary array is used which contains a certain number of different DNA molecules. The spot size, symmetry, distances, and quantity does not matter, as well as the Orientation of the DNA molecules on the surface.

Step 2:

To perform the following scan, you can choose between two different transmitter chips:

i) A transmitter chip which has only been coated with the primers required for the subsequent PCR.

ii) A transmitter chip in which 5 'of the primer sequence an additional DNA sequence of any length is present.

Furthermore, an additional DNA sequence can be introduced by adding it in solution, so that the entire transfer area is extended or changed by this DNA sequence.

Step 3:

The transmitter chip is now filled with fresh DNA mix and a template, which has homologous sequences to the already existing DNA molecules. This template is added in abundance, so that it can not impoverish in the subsequent fusion PCR. The cavity chip is now sealed with a non-functionalized surface and then a standard PCR is performed.

Step 4:

The stored information in the form of DNA molecules of the transmitter chip can be copied to a new, empty, specially functionalized surface by means of PCR and a standard PCR mix. c) ST subtraction

Step 1 :

It is used primary array, which contains a certain number of different DNA molecules. The spot size, symmetry, spacings and quantity are irrelevant, as well as the orientation of the DNA molecules on the surface. Step 2:

First, a scan of one of the primary arrays is made by PCR using a transmitter chip and a conventional PCR mix. In this case, the DNA of the primary array can be transferred by PCR into the transmitter chip by selecting the primer used in full length or partial length. The spatial information is completely preserved.

Step 3:

The stored information in the form of DNA molecules of the transmitter chip can be copied by PCR and a standard PCR mix to a new, empty, specially functionalized surface. d) RT Zoom

Step 1 :

It is used primary array, which contains a certain number of different DNA molecules. The spot size, symmetry, distances, and quantity are irrelevant, as well as the orientation of the DNA molecules on the surface.

Step 2:

First, a scan of one of the primary arrays is made by PCR using a transmitter chip and a conventional PCR mix. Here, the geometry and arrangement of the transmitter chip is chosen so that it is equal to that of the output microarray. As a result, a DNA point of the output array ever comes to lie under a single cavity. The cavity diameter is chosen so that the cavity is either larger or smaller than the DNA spot of the primary array. After completion of the PCR, the spatial and content information of the original DNA spot was transferred into each one cavity of the transmitter chip.

Step 3:

The stored information in the form of DNA molecules of the transmitter chip can be copied to a new, empty, specially functionalized surface by means of PCR and a standard PCR mix. e) RT rotation

Step 1 :

Step 3:

The transmitter chip is rotated by a certain angle and by a certain point. Step 4:

The stored information in the form of DNA molecules of the transmitter chip can be copied to a new, empty, specially functionalized surface by means of PCR and a standard PCR mix.

f) RT shift

Step 1 :

It is used primary array, which contains a certain number of different DNA molecules. The spot size, symmetry, spacings and quantity are irrelevant, as well as the orientation of the DNA molecules on the surface.

Step 2:

First, a scan of one of the primary arrays is made by PCR using a transmitter chip and a conventional PCR mix. After completion of the PCR, the same DNA species are present in the transmitter chip, which are also present on the output DNA microarray. Their spatial arrangement has also been preserved. As a result, both the spatial, as well as the content information of the primary array was thus transferred to the Überträgerchip.

Step 3:

The transmitter chip is shifted by a certain length in the x or y direction.

Step 4:

The stored information in the form of DNA molecules of the transmitter chip can be copied by PCR and a standard PCR mix to a new, empty, specially functionalized surface. g) RT merge

Step 1 :

Step 2:

First, a scan of one of the primary arrays is made by PCR using a transmitter chip and a conventional PCR mix. Here, the structure of the transmitter chip is chosen so that any number of points of the primary array is transferred to the same structure of the transmitter chip. The biomolecules are not fused thereby, but merely mixed in the same structure. After completion of the PCR are the

Mixtures or individual DNA species in the structures of the transmitter chip.

Step 3:

The stored information in the form of DNA molecules of the transmitter chip can be copied by PCR and a standard PCR mix to a new, empty, specially functionalized surface. The resulting secondary array now consists of mixed spots. Individual monoclonal spots may also be present depending on the choice of the structure of the transmitter chip. h) RT resolution

Step 1 :

Step 2:

First, a scan of one of the primary arrays is made by means of a transmitter chip and a conventional PCR mix by means of PCR. Here, the structure of the transmitter chip is chosen so that either more or fewer structures compared to the primary array are available. After completion of the PCR, then, e.g. transfer a spot of the primary array into many smaller structures of the transmitter chip (higher resolution), or transfer multiple spots of the primary array into a larger structure of the transmitter chip (lower resolution)

Step 3:

The stored information in the form of DNA molecules of the transmitter chip can be copied by PCR and a standard PCR mix to a new, empty, specially functionalized surface. The resulting secondary array now consists of more or fewer spots compared to the primary array. i) FT

Step 1 :

Step 2:

First, a scan of one of the primary arrays is made by PCR using a transmitter chip and a conventional PCR mix. Here, the structure of the transmitter chip is chosen to have a different shape than the spots of the primary array (e.g., stars or elongated spots). After completion of the PCR, the same DNA species are in the

Transmitter chip, which are also present on the initial DNA microarray. Their spatial arrangement has also been preserved. As a result, both the spatial, as well as the content information of the primary array was thus transferred to the Überträgerchip.

Step 3:

The stored information in the form of DNA molecules of the transmitter chip can be copied by PCR and a standard PCR mix to a new, empty, specially functionalized surface. The resulting secondary array now consists of spots of different shape compared to the primary array.

Figure 1. Regressive transformation (big to small). An array of hexagons has been transformed into smaller circles. The illustration shows a section of 2 hexagons.

Figure 2. progressive transform (small to large) The array on the left was transformed into a large spot array (right) with much larger cavities. Depending on the exact location of the original spots different products can be produced. In the example of the lower box, the lower red dot (A) was transformed into a red circle (A) with a much larger diameter. The red dot (A) above, however, was fused with the overlying green dot (C) to a yellow circle (B). Directly above this box is a green spot (small on the left, large on the right), which in turn was transformed into a pure form. In the case of the box on the top left, 2 green (C) and 2 red spots (A) (left) were converted to a traffic light (red (A), yellow (B), green (C) (right)).

FIG. 3. Allocation of the transformation The data from FIG. 2 were superimposed here in order to render the transformation clearer. One sees very well, which small spots as germs of the have contributed to larger spots. Some of the small red spots did not produce any signal in this experiment. For the green points, there is a much clearer allocation.

Figures 4 to 17 show particularly preferred embodiments of the invention.

Figure 4: position optimization

Problem: Microarrays are printed, i. tiny drops come to a surface and are then different in size (A), not exactly in position (B) or inhomogeneous or have an irregular shape (C), etc. Automated capture of spots is difficult.

Solution: A transformation is performed that contains the optimal position.

FIG. 5: structure optimization

Problem: Microarrays are printed, i. the drops usually have roundish to oval shapes, now any structures can be created

Solution: A transformation is carried out which gives each sport an individual form, even those that can not yet be produced.

FIG. 6: Reduction 1

Problem: The format of arrays is determined by the printing process, i. The size of the spots is limited by the minimum delivery amount down, as well as their distance to avoid bleeding the spots

Solution: You can now create smaller spots in the transformation.

Figures 7 and 8: reduction 2a and 2b

Solution: Two transformations are performed, one that "pulls everything together" and then makes one the smaller spots.

FIG. 9: Enlargement 1

Problem: The format of arrays is determined by the printing process, i. the size of the spots is limited by the maximum delivery amount and concentration upwards.

Solution: larger spots can now be created in the transformation.

Figures 10, 11 and 12: magnification 2a, 2b and 2c

Problem: The format of arrays is determined by the printing process, ie the size of the spots is limited by the maximum output quantity and concentration. Solution: in a first transformation, the spots are spaced, then first reduced in size (!), To avoid contamination and then enlarged.

Figures 13 and 14: Position exchange a and b

Problem: The format of the array is fixed after printing, a position change automatically reprints.

Solution: In a first transformation, the positions are exchanged and the spot geometry is then restored in a second transformation.

Figure 15: Association

Problem: The DNA of individual spots can not be subsequently fused or mixed to allow for additive effects (for example, when two DNAs together first interact with a protein), or allosteric effects (when a DNA is needed to activate or activate a protein) which then binds to another DNA, eg, initiation sequences or the lac promoter or lac repressor).

Solution: Two or more spots can be fused and the DNA mixed either side by side, or linearly one after the other.

FIG. 16: Splitting

Problem: Spots can not be resized later, so that a higher signal (in non-saturating conditions) or a faster kinetics (ambient analyte theory) can be achieved

Solution: In the transformation, the spot is split into smaller spots Figure 17: combinatorial mixture

Problem: Producing many DNA spots, but carrying identical partial sequences, is just as costly as generating completely disjointed sequences

Solution: The DNA is first taken from a template 1, then from a template 2, to then produce as a result a combinatorial mixture. It is thus possible to generate a total of n ^* m combinatorial mixtures with n and m spots on the original arrays.

A mixture can be both a juxtaposition (several DNA sequences are side by side on the surface) as well as an Aneinander (the DNA sequences are linearly consecutively attached to each other so extended) realized. This can be achieved by choosing the primers and DNA sequences during the amplification step of the biochemistry (all primers identical = mostly next to each other and primers matched = mostly together = usually extension PCR).

Figures 18 to 22 show particularly preferred devices of the invention. FIG. 23 shows a schematic representation of a typical transformation process.

Shown is the transformation of a small spot in a larger area spot as well as the transformation of a spatially large spot in 3 small spots a: filling the

Cavity chips with a reaction mix, e.g. PCR mix or cell-free expression system and placement of the original array on the cavity chip b: Performing a copying reaction, e.g. a PCR. c: opening, washing and blocking of the chip d: filling of the cavity chip with reaction mix and sealing it with an empty array surface. e: performing a copying reaction, e.g. a PCR. f: washing and blocking of the newly formed array (transformed array) and the cavity chip.

Figure 24 shows another example of a possible application.

Abbreviations and explanations:

Transmitter chip Also called cavity chip; Device used for local

Storage of biomolecules can be used

Primary array Also called template array; Microarray, which serves as Ausganslage and was produced according to the current state of the art; preferably DNA or RNA array

Secondary array microarray, which is done after an array

Transformation is made. It represents the end result of the array transformation.

RT space transformation - changing the information of the location and / or the geometry compared to the primary array

RT Zoom Room transformation Zoom - The location information remains; The

Spots are displayed enlarged or reduced.

RT Rotation Space transformation Rotation - Rotation of the primary array

specific point and angle (in addition to displacement)

RT shift spatial transformation displacement - change of position of the array in the

Comparison to the output array

RT stretching Redundancy to shape transformation

RT Add or connect new information already

Merging existing information. The texture of the original ones

Information is not changed here. RT resolution space transformation resolution change increase or

Reduction of the number of spots compared to the output array

FT Form Transformation - Changing the Original Form of the

Arraypoints of the primary array

ST sequence transformation - modification of biomolecules compared to

primary array

(additive) ST addition Sequence transformation that allows the addition of new information to the already existing information

(subtractive) ST Sequence transformation that allows the partial or complete removal of subtraction of information

According to the theory of affine and partially affine mappings from mathematics, all of the transformations used can be bijective, injective, and / or surjective and, in addition, additive, subtractive, or identical. Where:

Bijective: Each cavity of the transmitter chip hits exactly one point of the primary array. No points are left out.

Injection: The cavities of the transmitter chip hit one point of the primary array. Points can be omitted

Surjective: Each cavity of the transmitter chip hits one or more points of the primary array. No points are left out.

credentials

1. Kishawi, Iman: Agilent Array Technology and Custom Capabilities, Agilent Technologies, October 2008, pages 1-53

2. LeProust, Emily: Agilent's Microarray Platform: How High-Fidelity DNA Synthesis Maximizes the Dynamic Range of Gene Expression Measurements, Agilent Technologies, December 21, 2015, pages 1-12

3. Blanchard, A.P., Kaiser, R.J .; Hood, L.E .: High density oligonucleotide arrays, biosensors &

Bioelectronics Vol. 11, no. 6/7, 1996, pages 687-690

4. Lin, H., Sun, L., Crooks, R.M .: Replication of a DNA Microarray, CHEM. SOC. 9 VOL. 127, NO. 32, 23 July 2005, pages 11210-11211

5. Kim, J., Crooks, R.M .: Parallel Fabrication of RNA Microarrays by Mechanical Transfer from a DNA Master, Anal. Chem., 27 October 2007, pages 8994-8999

6. Kim, J., Crooks, R.M .: Replication of DNA Microarrays Prepared by In Situ Oligonucleotides

Polymerization and Mechanical Transfer, Analytical Chemistry Vol. 79, No.19, 1 October 2007, pages 7267-7274

7. Lin, H. et al .: Replication of DNA Microarrays from Zip Code Masters, CHEM. SOC. Vol. 128, no.

10, 18 February 2006, pages 3268-3272

8. De Risi, J., et al .: Use of a cDNA microarray to analyze gene expression patterns in human cancer, Nat. Genet., December 1996, pages 457-460 Nuwaysir, EF, el.: Gene Expression Analysis Using Oligonucleotide Arrays Produced by Maskless Photolithography, Genome Research, 16 April 2002, pages 1749-1755

Pease, A.C., et al. : Light-generated oligonucleotide arrays for rapid DNA sequence analysis, Proc. Natl. Acad. Be. USA Vol. 91 Biochemistry, May 1994, pp. 5022-5026

Yu, A. Amy, et al. : Supramolecular Nanostamping: Using DNA as Movable Type, Nano Letters 2005 Vol. 6, 14 March 2005, pages 1061-1064

Yu, A., Stellacci, F .: Contact Printing Beyond Surface Roughness: Liquid Supramolecular

Nanostamping, Advanced Materials, 2007, pages 4338-4342

Claims

claims

1. A method of microarray transformation comprising the following steps a) providing a template array, wherein the template array comprises a plurality of spots with template molecules and wherein the template molecules are preferably oligonucleotides, b) providing a cavity chip comprising a transfer matrix, c) providing a reaction mixture in the cavity chip d) placement of the precursor array on the cavity chip, e) copying process, wherein the oligonucleotides of the spots of the precursor array are copied onto the cavity chip f) provision of an array surface. g) providing a reaction mix in the cavity chip, h) placing the array surface on the cavity chip, i) copying process, whereby the oligonucleotides of the spots of the cavity chip are copied onto the array surface as DNA, RNA or protein and wherein this newly formed further array is in terms of spot shape, Spot size, spot location and / or in terms of containing information differs from the template array.

2. The method of claim 1, wherein a modification, extension, shortening,

Derivatization and / or inversion of the molecules in the cavities of the cavity chip and / or takes place during the copying processes.

3. The method of claim 1 or 2, wherein the steps a) to e) are repeated at least once, wherein the same cavity chip but another template array or the same template array is used in another or the same orientation.

4. The method according to at least one of the preceding claims, wherein the copying step comprises an amplification step.

5. The method according to at least one of the preceding claims, wherein the reaction mix a PCR mix, an isothermal amplification mix, a reverse transcription mix, a

Transcription mix or a cell-free expression mix.

6. The method according to at least one of the preceding claims, wherein the template molecules are oligonucleotides, preferably DNA molecules or RNA molecules.

7. The method according to at least one of the preceding claims, wherein the cavities of the cavity chip are coated with primers.

8. The method according to the preceding claim, wherein the primers at the 3 'end or at the 5' end carry an additional DNA sequence.

9. The method according to at least one of the preceding claims, wherein a plurality of spots of the template array are combined in the cavity chip and / or further array so as to produce a mixture of template molecules or the molecules derived therefrom and / or to generate a DNA or RNA which comprises several partial sequences or derivatives of these partial sequences of the template molecules from the respective spots.

10. The method according to at least one of the preceding claims, wherein spots of

at least two template arrays are merged in the further array so as to create a mixture of copies and / or to produce a copy comprising a plurality of subsequences of these subsequences of the template molecules from the respective spots.

1 1. Method according to at least one of the preceding claims, wherein at least one spot of the template array in the further array is subdivided into a plurality of spots.

12. The method according to at least one of the preceding claims, wherein additional DNA molecules are added in solution, so that the molecules are extended or changed by this DNA sequence.

13. The method according to at least one of the preceding claims, wherein the

Template molecules are DNA molecules which all or partially have short, identical DNA sequences of preferably 10-30 base pairs. ,

14. The method according to claim 8 or 13, wherein the additional sequences can be used as a barcode for location information and / or the primer is a sequence for

Transcription and / or cell-free synthesis may contain

15. The method according to at least one of the preceding claims, wherein the

Transformation is spatially and / or temporally limited.

16 cavity chip for performing a method according to at least one of the preceding claims, comprising a transfer matrix with cavities, wherein the cavities are arranged so that a transformation can take place.

17. A device for carrying out a transformation according to at least one of the preceding claims, comprising

A cavity chip according to the preceding claim,

• at least one template array and

• at least one array surface.